The present invention is in the field of transporter cofactor proteins that are related to the sodium/hydrogen exchanger-regulatory factor (NHE-RF) subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect ligand transport and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.
The novel human protein provided by the present invention is related to transporter regulatory factors, also referred to as transporter cofactors. In particular, the protein of the present invention is similar to regulatory factors of sodium/hydrogen exchangers (solute carrier family 9), referred to as sodium/hydrogen exchanger-regulatory factors (NHE-RFs). NHE-RFs participate in the protein kinase A regulation of sodium/hydrogen exchangers, such as those found at the renal brush border, and are typically found in actin-rich structures (Reczek et al., J Cell Biol Oct. 6, 1997; 139(1):169-79; Weinman et al., J Clin Invest 1995 May;95(5):2143-9). Furthermore, the protein of the present invention shows a high degree of similarity to ezrin-radixin-moesin (ERM) proteins, which are related to the NHE-RF family, particularly ERM-binding phosphoprotein 50 protein (EBP50). The ERM family of proteins are membrane-cytoskeletal linking proteins that have NH2- and COOH-terminal domains that bind the plasma membrane and the actin cytoskeleton, respectively (Reczek et al, J Cell Biol Oct. 6, 1997; 139(1):169-79).
ERM proteins are found in high concentrations in the apical portion of polarized microvilli-containing epithelial cells, which are abundant in such locations as the placenta and intestinal brush border; the actin filaments within microvilli must attach to the epithelial cell membranes in order to properly assemble and maintain the microvilli. ERM proteins are involved in linking integral membrane proteins and cytoskeletal proteins such as actin. Actin cytoskeletal assembly requires activation of sodium/hydrogen exchangers, which is modulated by NHE-RF proteins.
EBP50 binding interactions are involved in such important biological processes as membrane protein trafficking, regulating the activity of interacting proteins, and moving such interactors into common microdomains to facilitate interaction .(Fouassier et al., J Biol Chem Aug. 11, 2000; 275(32):25039-45). Additionally, a human homologue of NHE-RF has been identified as an interactor of merlin, which is a neurofibromatosis 2 tumor suppressor protein; the human NHE-RF may play a critical role in the tumor suppressor functions of merlin (Murthy et al., J Biol Chem Jan. 16, 1998; 273(3):1273-6).
EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains, which are known to bind integral membrane proteins (Reczek et al., J Cell Biol Oct. 6, 1997; 139(1):169-79). NHE-RF modulates protein kinase A regulation of sodium-hydrogen exchanger through binding of the PDZ domains. NHE-RF may also bind to the ERM proteins, ezrin, moesin and radixin, via the PDZ domains (Murthy et al., J Biol Chem Jan. 16, 1998; 273(3):1273-6). Furthermore, EBP50 proteins self-associate with other EBP50 proteins via their PDZ domains, providing enhanced functional capabilities, such as the ability to form multiprotein complexes and regulate membrane transport processes (Fouassier et al., J Biol Chem Aug. 11, 2000; 275(32):25039-45).
Transporter cofactors, particularly members of the sodium/hydrogen exchanger-regulatory factor subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins.
The present invention is based in part on the identification of amino acid sequences of human transporter cofactor peptides and proteins that are related to the sodium/hydrogen exchanger-regulatory factor subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter cofactor activity in cells and tissues that express the transporter cofactor. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt""s lymphoma, and leukocytes.